A scanner head which is, however, not suitable for high-resolution scanning fluorescence microscopy is known from WO 90/00754 A1. The known scanner head comprises a lens which shapes excitation light emerging from a fiber optical waveguide into a parallel light beam. The light beam is deflected by two mirrors tiltable by drives and directed onto a sample via an ocular mount of a light microscope through the objective lens of the light microscope. For scanning the sample with the light beam focused by the objective lens, the mirrors are tilted. The fluorescence light emerging from the ocular in the opposite direction is directed back onto the lens by the mirrors and the lens injects the light into the fiber optical waveguide. The fiber optical waveguide branches off in the direction of a light source for providing the excitation light one the one hand and to a detector for registering the fluorescence light on the other hand. The light source and the detector therefore are not part of the scanner head but are connected to the latter through the fiber optical waveguide. A control for the drives of the mirrors is also provided outside of the scanner head.
A laser scanning microscope in known from DE 197 02 753 A1, corresponding to U.S. Pat. No. 6,167,173 A and U.S. Pat. No. 6,486,458 B1, which comprises an arrangement for injecting laser radiation into a scanner head with a scanner deflecting at least in two dimensions. The radiation is focused into a sample through a light microscope. The injection of the radiation is achieved through at least one fiber optical waveguide, while collimation optics for collimation of the divergently emerging radiation is provided downstream of the fiber end at the scanner head. Seen from the direction of the sample, downstream of the scanner in the scanner head a detector for detecting the radiation from the scanned object is provided.
A high-resolution scanning fluorescence microscope and a module for a high-resolution scanning fluorescence microscope are known from DE 101 05 391 A1, corresponding to U.S. Pat. No. 6,958,470 B2. The high-resolution scanning fluorescence microscope comprises a light source for emitting an excitation light beam suitable for exciting an energy state of the sample, a detector for detecting emission light and a stimulating light beam coming from the light source for causing stimulated emission in the sample excited by the excitation light beam. The excitation light beam and the stimulating light beam are arranged in such a way that their intensity distributions partly overlap in a focal region. Optical elements shaping the stimulating light beam are combined into at least one module which may be positioned in the beam path of the scanning fluorescence microscope. In practical terms, the module may have a bayonet mount with which it is connectable to a corresponding mount of the scanning fluorescence microscope. With the known module for a high-resolution scanning fluorescence microscope, existing scanning fluorescence microscopes are intended to be upgraded to STED microscopes. The adjustment of the optical elements shaping the stimulating light beam with respect to the scanning fluorescence microscope to reach a full function of the scanning fluorescence microscope as an STED microscope, however, proves to be difficult. The stimulating light beam only then has its intended intensity distribution in the focal region if it is aligned exactly with respect to a pupil of the objective lens of the scanning fluorescence microscope and if it keeps this alignment when scanning the sample with the scanner of the scanning fluorescence microscope.
Under the heading of “easySTED”, high-resolution scanning fluorescence microscopes are known in which the excitation light and the fluorescence inhibition light together pass through beam-shaping optics which, however, has a different effect on the excitation light and the fluorescence inhibition light. Especially, the fluorescence inhibition light is shaped in such a way that it comprises an intensity minimum surrounded by intensity maxima at the intensity maximum of the excitation light focused in a diffraction-limited spot. Examples for beam-shaping optics which come under the heading of “easySTED” are described in DE 10 2007 025 688 A1, corresponding to U.S. Pat. No. 8,755,116 B2, WO 2010/133678 A1 and DE 10 2014 113 716 A1.
From EP 2 359 178 A1, corresponding to U.S. Pat. No. 8,520,280 B2, a device for dynamic shift of a light beam with respect to optics focusing the light beam and comprising a pupil is known. The dynamic shift is accomplished in order to scan an object in a two-dimensional scanning area with the focused light beam. The device has beam deflectors, which deflect the light beam in two different directions with respect to the optical axis of the optics by dynamically adjustable deflection angles. For each direction at least two beam deflectors are series-connected and may be controlled independently of each other so that the beam position of the light beam in the respective direction within the pupil of the focusing optics as well as the angle of the light beam with respect to the optical axis of the focusing optics and therefore the position of the focused light beam in the sample region may be set. In this way it becomes possible to scan the two-dimensional scanning region without variation of the optical conditions above the scanning region. In this way it is e. g. prevented that the spatial phase structure of the stimulating beam shifts within the pupil during STED microscopy. Such shifts lead to the desired light intensity distribution of the focused stimulating beam not having the low intensity minimum surrounded by high intensity maxima at the location of the intensity maximum of the excitation light beam. Furthermore, with the known device distances of the deflectors to the pupil or of a pupil image of the focusing optics as well as lens aberrations of the focusing objects and aberrations of the beam deflectors may be compensated. Furthermore, the pivoting point of the deflected light beam may be set in an axial direction in order to e.g. provide for different axial positions of different objective lenses.
The scientific publication “Stimulated Emission Depletion microscopy to study a myloid fibril formation” (P. Mahou et al., Single Molecule Spectroscopy and Superresolution Imaging VIII, J. Enderlein et al. (ed.), Proc. of SPIE Vol. 9331, 2015) discloses a “home-built” STED microscope using a light microscope and an optical setup external to the light microscope. The optical setup includes a laser light source emitting a light beam. The light beam is split into an excitation beam and a depletion beam. The excitation beam is fed through a light path including 30 m (98 ft) of optical fiber. The depletion beam is fed through a separate light path including 100 m (328 ft) of optical fiber, in the course of which it is shaped into a “doughnut” shape, so as to provide stimulated emission depletion to prevent fluorescence emission. Through a first dichroitic mirror, both light beams are employed to scan a sample using an objective lens of the light microscope and a quad scanner comprising four tilting mirrors. The fluorescence light from the sample is fed back through the light microscope objective lens, the quad scanner and the first dichroic mirror before being deflected onto a detector by a second dichroic mirror.
There still is a need of a scanner head by which existing light microscopes of different types and different manufacturers can be upgraded to high-resolution scanning fluorescence microscopes without problems.